Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cytotoxic and cell kinetic effects of the epipodophyllotoxin 4,6-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyr anoside) (VP-16) in cultured mammalian cells are thought to relate to the induction of DNA damage, specifically DNA strand interruptions. In an effort to explore this relationship in human cells we have identified a VP-16-hypersensitive human cell system, namely an SV40-transformed fibroblast line (AT5BIVA) originally derived from an ataxia telangiectasia (AT) patient. Evidence is presented that enhanced VP-16 sensitivity may be a consistent in vitro feature at AT derived cells. However, the intrinsic sensitivity (DNA strand breaks per lethal hit quantitated by nucleoid sedimentation) was the same for AT5BIVA and a corresponding normal control, indicating that the AT cell line accumulated more drug-induced DNA damage during short-term VP-16 exposures. It is suggested that AT cells may have abnormal topoisomerase II activity. The cell cycle responses of normal and AT cells to VP-16 exposure were complex, with the generation of parasynchronous S phase populations and the accumulation of cells in G2. Differences in cell killing or DNA strand breakage between normal and AT cells could only be correlated with the magnitude and kinetics of the G2 retention phenomenon. In short, there are several similarities in the action of ionizing radiation and VP-16. We suggest that the sensitivity of cellular DNA to VP-16-induced DNA damage and the kinetics of the G2 delay may be useful parameters for predicting the survival probability of drug-treated human tumor populations.
 ...
PMID:Predominant role for DNA damage in etoposide-induced cytotoxicity and cell cycle perturbation in human SV40-transformed fibroblasts. 301 31

It has been recently shown that VP-16-213, a semi-synthetic derivative of podophyllotoxin, inhibits the function of mammalian DNA topoisomerase II. In the present study, we examined the effects of VP-16-213 on the replication of herpes simplex virus type 2 (HSV-2). The compound did not inhibit the synthesis of early viral polypeptides at concentrations at which viral DNA synthesis was strongly suppressed, but induced double-strand breaks in newly synthesized HSV DNA. Electron microscopic examination of treated cells revealed the presence of a number of capsids with empty or partial cores. The level of topoisomerase II activity remained unaltered after infection, and all attempts to isolate VP-16-213-resistant mutants of HSV-2 have failed in spite of extensive efforts. It is suggested therefore that the mode of action of VP-16-213 may be inhibition of viral DNA synthesis by impairing the function of host cell topoisomerase II.
 ...
PMID:Effects of the epipodophyllotoxin VP-16-213 on herpes simplex virus type 2 replication. 302 14

The DNA intercalator, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and the nonintercalator, etoposide (VP-16) produce topoisomerase II-mediated protein-linked DNA strand breaks. This function of topoisomerase II was investigated in relation to cell proliferation and cell cycle. Mouse fibroblasts NIH 3T3 and mouse leukemia L1210 cells stop proliferation when they reach a certain density. Nuclei were isolated from proliferative or quiescent cells and then treated with drug for 30 min. DNA modifications were assayed by alkaline elution. We found that the frequencies of m-AMSA- or VP-16-induced DNA-protein links were higher in nuclei from exponentially growing than in those from quiescent cells in both the 3T3 and the L1210 lines. Drug-induced protein-associated DNA breaks were also studied as a function of the cell cycle in 3T3 cells that had been arrested by contact inhibition in medium containing 1% calf serum and then stimulated to proliferate by raplating at a lower cell density in medium containing 10% serum. In these synchronized cells, a large peak of [3H]thymidine incorporation occurred 18-30 h after replating. The yield of DNA-protein cross-links produced by 30-min drug treatments of nuclei isolated at various times after growth initiation increased concomitantly with the peak of the DNA synthesis. The topoisomerase II activity of nuclear extracts, as measured by kinetoplast DNA decatenation followed a similar pattern. Using colony-forming assays, we also observed that m-AMSA and VP-16 were most cytotoxic in proliferative cells and during DNA synthesis. These results suggest that alkaline elution measurement of m-AMSA- or VP-16-induced protein-linked DNA breaks reflects the association of topoisomerase II with DNA. This association is increased during DNA replication, making the cells more vulnerable to m-AMSA and VP-16 at this time.
 ...
PMID:Topoisomerase II-mediated DNA breaks and cytotoxicity in relation to cell proliferation and the cell cycle in NIH 3T3 fibroblasts and L1210 leukemia cells. 303 May 40

We have examined DNA in cells treated with 5,6-dichloro-1-beta-O-ribofuranosylbenzimidazole (DRB), an adenosine analogue. The results show that DRB induces an partial fragmentation of DNA when the cells are lysed in dilute alkali. Fragmentation of DNA does not occur in control cells, nor in cells pretreated with novobiocin or VP-16/VM-26. The data show that DRB interferes with DNA topoisomerase II. In agreement with this interpretation, crude nuclear extracts of DRB-treated cells result in reduced in vitro KC1/SDS precipitation of covalent protein-DNA complexes. Formation of covalent complexes is typical of topoisomerase-DNA interaction.
 ...
PMID:5,6-Dichloro-1-beta-O-ribofuranosylbenzimidazole induces DNA damage by interfering with DNA topoisomerase II. 303 22

The presumptive intracellular target of the anti-leukemia agents 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and 4-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16) is the enzyme topoisomerase II. We found that 350 mM NaCl extracts of nuclei from HL-60 and HL-60/AMSA, an m-AMSA resistant HL-60 subline, contained equivalent topoisomerase II activity. However, the ability of m-AMSA to stimulate cleavage of exogenous DNA and to stimulate crosslinking of exogenous DNA with protein, processes which are topoisomerase II-mediated, was greatly reduced in the HL-60/AMSA extracts compared to the HL-60 extracts. HL-60 and HL-60/AMSA were almost equally sensitive to the cytotoxic effects of VP-16 and differences in VP-16-stimulated, topoisomerase II-mediated exogenous DNA cleavage and protein crosslinking between HL-60 and HL-60/AMSA extracts were much less than the differences in m-AMSA-stimulated exogenous DNA cleavage and protein crosslinking. Thus, the interaction between topoisomerase II activity, exogenous DNA, and m-AMSA or VP-16 indicated the susceptibility HL-60 and HL-60/ AMSA to the cytotoxic effects of the drugs. A similar correlation may exist in explanted leukemia cells from patients with acute myelogenous leukemia.
 ...
PMID:The interaction between nuclear topoisomerase II activity from human leukemia cells, exogenous DNA, and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) or 4-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16) indicates the sensitivity of the cells to the drugs. 303 64

The two demethylepipodophyllotoxin glycosides, teniposide (VM-26) and etoposide (VP-16), have previously been reported to interact with DNA topoisomerase II by stabilizing a topoisomerase II-DNA covalent intermediate. This study examined the protein-associated aspect of the topoisomerase II-DNA-epipodophyllotoxin lesion. We found that in mouse (L1210) and human (VA-13 and HT-29) log-phase cell cultures, all DNA strand breaks produced by VP-16 or VM-26 were protein-associated. We found also that these protein-associated breaks occurred with a frequency which correlated with cytotoxicity in all three cell lines. For all three cell lines and for both compounds the regression lines were similar. Therefore, for a given class of topoisomerase II inhibitors, it may be possible to generate a characteristic line from which DNA-protein crosslink frequency predicts cytotoxicity.
 ...
PMID:Protein-linked DNA strand breaks produced by etoposide and teniposide in mouse L1210 and human VA-13 and HT-29 cell lines: relationship to cytotoxicity. 304 Dec 38

A new DNA precipitation assay used together with the alkali unwinding assay may provide a rapid means of detecting DNA damage in addition to strand breaks based on the relative amount of damage measured by the two assays. X-rays, Adriamycin, 4-nitroquinoline-N-oxide, N-methyl-N'-nitrosoguanidine, bleomycin, RSU 1172, and five other drugs produced the same relative amount of strand breakage by using the DNA precipitation and alkali unwinding assays. However, strand breaks produced by the bifunctional alkylating agents bis(2-chloroethyl)nitrosourea, RSU 1069, and RSU 1131 were detected with greater efficiency by the DNA precipitation assay, while the unwinding assay measured more strand breaks than the precipitation assay after damage by the topoisomerase inhibitors VP-16 and VM-26 and the DNA-condensing agents acridine orange and pyronin Y. Based on the reported mechanisms of action of these drugs, and studies with known DNA cross-linking agents, it appears that in addition to DNA strand breaks, the alkali unwinding assay is more sensitive to interstrand than to DNA-protein cross-links, while the DNA precipitation assay can be used to detect both types of cross-links. While quantification of specific lesions is not possible with this approach, the concomitant use of these two assays may provide a rapid and simple method for screening genotoxic drugs for DNA damage, and may also help to differentiate between DNA lesions which include strand breaks, interstrand and protein cross-links, DNA-phosphate adducts, and DNA-drug precipitates.
 ...
PMID:Comparison between the DNA precipitation and alkali unwinding assays for detecting DNA strand breaks and cross-links. 318 60

Many intercalative antitumor drugs have been shown to cleave DNA indirectly through their specific effect on the stabilization of a cleavable complex formed between mammalian DNA topoisomerase II and DNA (Nelson, E.M., Tewey, K.M., and Liu, L.F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1361-1365). Antitumor epipodophyllotoxins (VP-16 and VM-26) which do not intercalate DNA can similarly induce protein-linked DNA breaks in cultured mammalian cells. In vitro studies using purified mammalian DNA topoisomerase II show that epipodophyllotoxins interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by stabilizing a cleavable complex. Treatment of this stabilized cleavable complex with protein denaturants results in DNA strand breaks and the covalent linking of a topoisomerase subunit to the 5'-end of the broken DNA. Furthermore, epipodophyllotoxins also inhibit the strand-passing activity of mammalian DNA topoisomerase II, presumably as a result of drug-enzyme interaction. The agreement between the in vivo and in vitro studies suggests that mammalian DNA topoisomerase II is a drug target in vivo. The similarity between the effect of epipodophyllotoxins on mammalian DNA topoisomerase II and the effect of nalidixic acid on Escherichia coli DNA gyrase suggests that the cytotoxic action of epipodophyllotoxins may be analogous to the bactericidal action of nalidixic acid.
 ...
PMID:Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. 609 81

Epipodophyllotoxins are an important new class of anticancer agents which include the compounds VM-26 (teniposide) and VP-16 (etoposide). The mechanism of action of these drugs appears to involve production of DNA single- and double-strand breaks by virtue of a temperature-sensitive interaction between drug and a heat-labile intranuclear component. We now report evidence indicating that type II topoisomerase is the likely intracellular target for the DNA strand-breaking effects of the epipodophyllotoxins. Both VM-26 and VP-16 stimulate site-specific DNA cleavage by a highly purified calf thymus type II topoisomerase. VM-26 is 5- to 10-fold more potent than VP-16 in this assay, a difference that is also seen when DNA strand breaks are assayed in isolated nuclei of mouse leukemia cells following drug exposure. Furthermore, a similar potency difference exists with respect to cytotoxicity. Equilibrium dialysis experiments using [3H]VP-16 indicate that the drug does not bind to DNA. Thus, we suggest that the epipodophyllotoxins exert their anti-cancer effects by "poisoning" type II topoisomerase without binding to DNA. In this regard, their actions may be analogous to those of nalidixic acid in bacteria.
 ...
PMID:Role of topoisomerase II in mediating epipodophyllotoxin-induced DNA cleavage. 609 1

We selected and characterized a 30-fold etoposide (VP-16)-resistant subline of K562 human leukemia cells (K/VP.5) that exhibits quantitative and qualitative changes in topoisomerase II, including hypophosphorylation of this drug target. The initial rate of topoisomerase II phosphorylation was reduced 3-fold in K/VP.5 compared with K562 cells, but the rate of dephosphorylation was similar. Analysis of potential topoisomerase II protein kinases revealed a 3-fold reduction in the level of the beta II protein kinase C (PKC) in K/VP.5 cells, whereas levels of alpha- and epsilon PKC, casein kinase II, p42map kinase, and p34cdc2 kinase were comparable for both cell lines. The PKC activator, bryostatin 1, together with K562 nuclear extracts potentiated VP-16-induced topoisomerase II/DNA covalent complex formation in nuclei isolated from K/VP.5 cells but not from K562 cells. Bryostatin 1 effects were blocked by the PKC inhibitor 7-O-methyl-hydroxy-staurosporine. Bryostatin 1 also up-regulated topoisomerase II phosphorylation and potentiated VP-16 activity in intact K/VP.5 cells but had no enhancing effect in K562 cells. 4 beta-Phorbol-12,13-dibutyrate and 12-O-tetradecanoylphorbol-13-acetate did not potentiate VP-16-induced topoisomerase II/DNA complex formation in intact cells or in isolated K/VP.5 nuclei. Together, our results indicate that beta II PKC plays a role in modulating the VP-16-induced DNA binding activity of topoisomerase II in resistant K/VP.5 cells through a mechanism linked to phosphorylation of topoisomerase II.
 ...
PMID:Hypophosphorylation of topoisomerase II in etoposide (VP-16)-resistant human leukemia K562 cells associated with reduced levels of beta II protein kinase C. 747 9


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>